(1) Field of the Invention
The present invention relates to a process for producing a poly-3-hydroxy butyric acid (hereinafter referred to as PHB).
More particularly, the present invention is addressed to a single stage continuous fermentation process for the production of bacterial cells containing a poly-3-hydroxy butyric acid.
(2) Description of the Related Art
PHB is produced and accumulated as an energy storage substance in bacterial cells of a large number of microorganisms. Since PHB is a thermoplastic polymer exhibiting an excellent biodegradability and biocompatibility, it has attracted attention as a "clean" plastic which does not destroy environment.
Also, PHB has been expected for many years to apply in various fields including biomedical materials such as surgical threads and fracture braces, and gradually releasing systems where a drag or pesticide is gradually released to environment.
Particularly, since synthetic plastics have caused serious public problems from the standpoint of environmental pollution as well as resource circumstances in recent years, PHB has been watched with an interest as a biopolymer which does not rely upon petroleum.
Heretofore, several processes for producing PHB have been proposed in Unexamined Japanese Patent Publication Nos. 60-214888 and 60-251889, and Examined Japanese Patent Publication Nos. 02-20238 and 03-65154.
The Examined Japanese Patent Publication No. 02-20238 has disclosed a process for the production of PHB by continuously fermenting bacterial cells of a genus of Alcaliqenes by using glucose as a source of carbon (hereinafter may be referred to as substrate) under a condition of restricted growth by limiting the feeding of nitrogen or phosphorus.
The Unexamined Japanese Patent Publication No. 60-214888 and Examined Japanese Patent Publication No. 03-65154 have disclosed processes for producing PHB by fermenting bacterial cells of a genus of Azotobacter or Protomonas in a batch method under a condition of restricted growth similar to that used for the genus of Alcaliqenes.
However, these processes are unsatisfactory in commercial production of PHB due to their high production cost. Further, those processes have such a defect that the production cost of PHB can not be brought to a low level since a main nutrient used for growing bacterial cells, namely, a carbon source is expensive, that the accumulation of PHB by a continuous fermentation is insufficient, or that the production process is complicated since two stages fermentations are required.
The cost of a raw material (that is, a substrate) is an important factor in the total cost for the production of PHB.
While the processes for the production of PHB using glucose or saccharose as a raw material are described in the Examined Japanese Patent Publication No. 02-20238 and Unexamined Japanese Patent Publication No. 60-214888, the increase in the production cost of PHB can not be avoided in these processes.
A fermentation process by using inexpensive methanol as a substrate is described, for instance, in Unexamined Japanese Patent Publication No. 56-117793 where a microorganism of a species of Methylobacterium organophilum is continuously fermented using methanol as a substrate in a first fermentation vessel (fermenter) without the limitation of a nutrient. At this stage, the accumulation of PHB in cells will not be taken place. Then, the microorganism is continuously transferred to a second fermentation vessel in which the microorganism is subjected to further fermentation using nitrogen or phosphorus as a limiting factor for growth. At this stage, PHB is accumulated in cells for the first time. According to the description in the specification of Unexaminated Japanese Patent Publication No. 56-117793, however, the content of PHB is only 25 to 47% by weight based on the weight of dried cells.
The process where two stages fermentations are undertaken by using two fermentation vessels in a series has such defects that the process is complicated and that a satisfactory PHB content can not be obtained.
Further, while it is described in the Examined Japanese Patent Publication No. 02-20238 that a strain of Methylobacterium organophilum NCIB 11483 was continuously fermented with methanol as a substrate under nitrogen limitation, the maximum PHB content achieved under the conditions in the Publication No. 02-20238 was as low as about 11%.